2 tick case) after vaccination and a small expansion of circulating of plasmablasts in sequential samples. whole blood Interferon-Gamma-Release-immuno-Assay (IGRA) that uses two Qiagen? (Hilden, Germany) proprietary mixes of SARS-CoV-2 S-protein (Ag1 and Ag2) selected to activate both CD4 and CD8 T-cells, following manufacturer’s instructions. Briefly, venous blood samples were collected directly into the Quantiferon? tubes containing spike peptides as well as positive and negative controls. Whole blood was incubated at 37?C for 16C24?h and centrifuged to separate plasma. IFN- (IU/ml) was measured in these plasma samples in parallel using CLIA (Liason, Quantiferon? Gold Plus) and ELISA (QuantiFERON? Human IFN- SARS-CoV-2, Qiagen?) tests, both for research only use. Complete blood Count (CBC), flow cytometry lymphocyte phenotype and immunoglobulin levels were obtained in parallel samples.5, 6 Statistical analysis Data were analyzed by non-parametric tests; MannCWhitney test for comparisons between the COVID and NO-COVID groups, and Friedman test for comparisons between paired values, and correlation WRG-28 studies to compare variables was calculated by Pearson correlation coefficient. GraphPad Prism v8.01 software was used for both statistical analysis WRG-28 and graphical representation. Results In the COVID group plateau IgG (S) antibody levels were attained with first vaccination dose At enrolment COVID group participants (in the order of 0.2) (Fig. 4 ) but there was a good concordance in detecting the immune response to Spike. Sensitivity of the IGRA test may be superior in this group to IgG(S) antibody test due to the presence of one anti-CD20 treated participant (observe below, illustrative case 12), but due to the small size, conclusions cannot be drawn. Open in a separate windowpane Fig. 4 Correlation between the antibody and T cell reactions F2R with different IFN-g detection methods ELISA (A and C) and CLIA (B and D). Correlation between the T cell response to Ag1 and Ag2 with ELISA (E) and CLIA (F). One vaccine dose restores T cell response in post COVID participants, but vaccination boost was required for na?ve participants to attain a good response Interestingly four of the five post COVID group did not respond in the IGRA test prior to vaccination, but 1 dose was sufficient to reach good levels of IFN- production, indicating priming from the organic infection. Second dose rather reduced the response, maybe because due to the time elapsed since priming, response contracted more quickly, this reduction has now been reported in a recent paper of Lozano Ojalvo et al.7 and in additional not yet peer reviewed reports.8 In the NO-COVID group even if the first dose already induced a significant response, the boost vaccination was required to reach a response like that of the COVID group after first dose. In this small study, we did not find any connection of antibody nor T-cell reactions to spike proteins with the total lymphocytes, their main subsets, or the immunoglobulin levels. There was WRG-28 a tendency for antibody and T-cell reactions to be lower in older individuals (Fig. 5 ), no gender effect was detected. Open in a separate windowpane Fig. 5 Correlation between age and T cell reactions with different IFN-g detection methods ELISA (A, C, E and G) and CLIA (B, D, F and H) with Ag1 and Ag2; after vaccine (A, B, C and D) and after (vaccine) boost (E, F, G and H). Illustrative HCW instances As mentioned above one of the NO-COVID individuals, case 12, was treated with anti-CD20 for an autoimmune condition and did not develop IgG or IgM antibodies after vaccination. Interestingly a clearly measurable T-cell reactions was recognized (observe Fig. 2 tick case) after vaccination and a small development of circulating of plasmablasts in sequential samples. We recognized plasmablasts after the vaccine boost with few pre class switch B lymphocytes prior to vaccination despite anti-CD20 treatment, indicating residual capacity of the B-cell compartment (Fig. 6 ). Open in a separate windowpane Fig. 6 Analysis of naive B lymphocyte subpopulations in patient 12. Class switch, IgM WRG-28 memory space B cell populations and plasmablasts before vaccination (A), after 1st dose (B) and second vaccination dose (C) Memory space/switch B-lymphocytes were already present before vaccination and plasmablasts appeared only after vaccination. Another NO-COVID group patient, case 19, was infected the same day time that she received 1st dose of vaccination. The patient formulated T-cell response in the IGRA-assay, and in this case, in addition to antibodies to the S antigen, she formulated antibodies to N-antigen (Fig. 1A,.